The degeneration of the human retina, either as a result of trauma, age or disease, can result in permanent visual loss and affect millions of people worldwide. Degenerative conditions include, for instance, retinitis pigmentosa, age-related macular degeneration and diabetic retinopathy. These conditions are characterized by the progressive death of light sensing photoreceptor cells of the retina, and are the leading causes of incurable blindness in the western world. As the intrinsic regenerative capacity of the human retina is extremely limited, the only viable treatment option for people suffering from photoreceptor cell loss is cellular replacement.
Several cell types have been suggested as cell therapy tools for retinal degenerative disorders. Such cell types include human retinal progenitor cells (hRPC), retinal pigment epithelial cells, glial progenitors, neural stem cells, bone marrow and cord blood derived cells. Multipotent stem cells (also variously referred to as progenitor cells, immature cells, precursor cells, undifferentiated cells or proliferative cells) are an active focus for transplantation and differentiation.
Human retinal progenitor cells have great potential for use in clinical applications, such as for transplantation into degenerative or diseased retinal hosts. Such cells are typically isolated from fetal retina in pre- and post-natal tissue by removing the ciliary marginal zone and the optic nerve to eliminate contamination. See, for instance, U.S. Pat. No. 7,514,259, the disclosure of which is incorporated by reference herein. These cells can be expanded in vitro, and are capable of migrating into and repopulating the retina, forming new functioning photoreceptors.
The use of live cells as a drug product in clinical applications such as for transplantation requires the comprehensive characterization of the cell population during the expansion of the host population and prior to injection of the cells in a human recipient. Such characterization is typically achieved by identification of specific markers expressed on the surface of the cell. The identification of specific markers, i.e. markers unique to the particular cell line, is also useful for the enrichment or depletion of the host population.
Various attempts have been made in the past to identify specific markers unique to different cell types. Cell populations which have been successfully characterized and identified in this way include hematopoietic stem cells, embryonic stem cells, neural stem cells and glial progenitor cells.
Carter et al., BMC Ophthalmology, 9:1, 2009, describes the use of the marker CD133 to purify adult human retinal cells. Retinal cell suspensions were derived from adult human post-mortem tissue, and were enriched with magnetic automated cell sorting. Cell purification of about 95% was demonstrated.
U.S. Pat. No. 6,468,794 describes the enrichment of neural stem cell and progenitor cell populations using monoclonal antibodies that bind to cell surface markers. U.S. Pat. No. 7,015,037 describes isolated multipotent stem cells that are surface negative for the markers CD44, CD45, HLA Class I and HLA Class II. See, also, U.S. Pat. Nos. 6,908,763, 7,749,754 and 7,781,179, all of the respective disclosures of which are incorporated by reference herein.
Yuan et al., Plos One, 6(3), e17540, March 2011, describes improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells, glia and neurons derived from pluripotent stem cells. Cell surface signatures or markers are identified on the cells to enable the isolation of the cells from heterogeneous differentiating cell populations by fluorescent activated cell sorting. In particular, the reference states that a population of neural stem cells has been successfully isolated by using fluorescence activated cell sorting (FACS) and the following markers: CD184+, CD271−, CD44− and CD24+. The disclosure of this publication is hereby incorporated by reference herein in its entirety.
In spite of various attempts to characterize different types of stem cells using cell-specific markers, surface markers specific for human retinal progenitor cells have not been identified. The successful identification of such markers would advance the technology by permitting the enrichment of cell populations, the elimination of less desirable cells from the culture, such as glial cells, neural cells and retinal pigment epithelial cells, and the ultimate selection of retinal progenitor cells highly expressing the markers of interest. The highly purified cell populations can then be cultured using techniques such as those described in U.S. patent application Ser. No. 13/160,002, filed Jun. 14, 2011, and manufactured into cell banks for the purposes of deriving cells that could be used in human transplantation for the treatment of degenerative diseases of the retina. The cell surface markers can represent markers that allow characterization of the cell product to be delivered to the patient, in partial fulfillment of the requirements by government regulatory agencies for defining cell product quality.
In view of the aforementioned, as well as the importance of human retinal progenitor cells for clinical evaluation and use, it will readily be appreciated that a need exists to improve the characterization, identification and purification of human retinal progenitor cells for transplantation and disease treatment. These and other objectives of the invention will be clear from the following description.